GATA3 Promotes Ferroptosis Resistance by Repressing Integrin ß1 Signaling

Understanding mechanisms that determine the response of cells to ferroptotic stress is a timely issue that has significant ramifications for biology and pathology. We investigated these mechanisms in the context of breast cancer where tumors are comprised of diverse populations of cancer cells that differ in their ferroptosis sensitivity. Using single-cell RNA-sequencing, we determined that cancer cell populations with luminal differentiation are more resistant to ferroptosis than other cells within a heterogeneous tumor. Subsequent bioinformatic analysis and experimentation revealed that GATA3, a transcription factor that promotes luminal differentiation, has a causal role in ferroptosis resistance in luminal breast cancer cells. In pursuit of the mechanism involved, we found that GATA3 represses the expression of integrin ß1 and its downstream signaling cascade. This observation led us to demonstrate that integrin ß1 signaling is necessary for sensitivity to ferroptosis in basal breast cancer cells because it regulates a FAK/ROCK pathway that sustains the expression of ACSL4, a lipid-modifying enzyme that is essential for ferroptosis. The repression of integrin ß1 by GATA3 inhibits this signaling pathway rendering cells ferroptosis resistant. Together, these data provide insight into mechanisms of ferroptosis sensitivity and resistance that are linked to the cell biology and signaling pathways of the diverse types of cells present in breast tumors. Overall design: patient derived organoids were incubated with for either 3 or 12 hours with either DMSO (1:1000) or IKE (10 µM) prior to dissociation and sample collection. Specifically, the culture medium was aspirated and Matrigel was digested with trypsin at 370C for 10 minutes. The samples were then washed 3X with cold PBS and dissociated for 45 minutes with Accumax (StemCell cat. 07921) at room temperature to generate a single cell suspension, which was passed through a 0.45µm filter to prevent cell clumping. Samples were frozen at -800C in cryopreservation medium (30% organoid media with 10% DMSO and 60% FBS). Samples were then processed by GeneWiz where they underwent MACS dead cell removal and quality control prior to being processed through the 10X Genomics Chromium 3' gene expression RNA-seq protocol and Illumina sequencing. Cells were sequenced at a depth of 6000 cells per sample and 100,000 genes per cell. Raw sequencing data were converted to FASTQ and aligned to a human reference transcript using Cell Ranger(48).

阐明决定细胞对铁死亡（ferroptosis）应激应答的机制，是当前生物学与病理学领域兼具时效性与重要学术价值的研究课题。本研究以乳腺癌为对象探讨该调控机制：乳腺癌肿瘤由铁死亡敏感性存在差异的多群异质性癌细胞构成。通过单细胞RNA测序（single-cell RNA-sequencing），我们发现异质性肿瘤中具备腔上皮分化表型的癌细胞群，相较于其他癌细胞群对铁死亡的抗性更强。后续生物信息学分析与实验验证表明，GATA3作为促进腔上皮分化的转录因子，在腔面型乳腺癌细胞的铁死亡抗性中发挥了因果性调控作用。为明确其中的分子机制，我们发现GATA3可抑制整合素β1（integrin ß1）及其下游信号级联的表达。基于该发现，我们进一步证实：整合素β1信号通路对基底型乳腺癌细胞的铁死亡敏感性不可或缺——该通路可调控FAK/ROCK信号轴，以维持ACSL4的表达；ACSL4是一类对铁死亡至关重要的脂修饰酶。GATA3对整合素β1的抑制作用可阻断该信号通路，进而使细胞获得铁死亡抗性。综上，本研究数据揭示了与乳腺癌肿瘤内异质性细胞的细胞生物学特性及信号通路相关的铁死亡敏感性与抗性机制。实验设计：患者来源类器官先分别以终浓度1:1000的二甲基亚砜（DMSO）或10 μM的铁死亡诱导剂IKE孵育3小时或12小时，随后进行细胞解离与样本收集。具体操作如下：吸弃培养液，用胰蛋白酶于37℃下消化Matrigel（基质胶）10分钟；随后将样本用预冷的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤3次，再以Accumax（StemCell公司，货号07921）于室温下解离45分钟以制备单细胞悬液，将悬液通过0.45 μm滤膜以避免细胞成团。将样本置于含30%类器官培养液、10% DMSO及60%胎牛血清（Fetal Bovine Serum, FBS）的冷冻保存液中，于-80℃冻存。随后将样本送至GeneWiz公司处理：先通过MACS（磁珠激活细胞分选，Magnetic Activated Cell Sorting）技术完成死细胞去除与质量控制，再经10X Genomics Chromium 3'基因表达RNA测序（RNA-seq）流程建库，并进行Illumina测序。本研究每个样本的测序覆盖细胞数为6000个，单个细胞的测序基因读取量约为100000个；原始测序数据被转换为FASTQ格式后，使用Cell Ranger软件（参考文献48）比对至人类参考转录组。